Research Paper Preparation, Characterization and In-vitro release of Piroxicam-loaded Solid Lipid Nanoparticles

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1 1136 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 3 October-December 2010 Research Paper Preparation, Characterization and In-vitro release of Piroxicam-loaded Solid Lipid Nanoparticles V. K. Verma 1 * and A. Ram 2 1 Dibrugarh University, India 2 SLT Institute of Pharmaceutical Science, Bilaspur, India International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 3 October - December 2010 ABSTRACT: Solid lipid nanoparticles (SLNs) of piroxicam where produced by solvent emulsification diffusion method in a solvent saturated system. The SLNs where composed of tripamitin lipid, polyvinyl alcohol (PVAL) stabilizer, and solvent ethyl acetate. All the formulation were subjected to particle size analysis, zeta potential, drug entrapment efficiency, percent drug loading determination and in-vitro release studies. The SLNs formed were nano-size range with maximum entrapment efficiency. Formulation with 435nm in particle size and 85% drug entrapment was subjected to scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for surface morphology, differential scanning calorimetry (DSC) for thermal analysis and short term stability studies. SEM and TEM confirm that the SLNs are nanometric size and circular in shape. The drug release behavior from SLNs suspension exhibited biphasic pattern with an initial burst and prolong release over 24 h. KEY WORDS: Solid lipid nanoparticles (SLNs); tripalmitin; polyvinyl alcohol; piroxicam; drug entrapment efficiency Introduction A solid lipid nanoparticle (SLNs), introduced in 1991, to combined advantage and disadvantages of other colloidal carrier have attracted in increased attention in recent years, and is regarded as an alternative carrier system to traditional colloidal system, such as emulsion, liposomes and polymeric microparticles and nanoparticles. Nanoparticles made from solid lipids are attracting major attention as novel colloidal drug carrier for intravenous application as they have been proposed as an alternative particulate carrier system (Muller and Lucks 1991; Muller et al.1993; Muller and Lucks 1996; Almeida et al. 1997; Zur Muhlen et al. 1998). The solid lipid nanoparticles (SLNs) are submicron colloidal carrier ( nm), which are composed of physiological lipid, dispersed in water or in an aqueous surfactant solution. SLNs as colloidal drug carrier combine the advantages of polymeric nanoparticles, fat emulsion and liposomes simultaneously and avoiding some of their disadvantages. In order to overcome the disadvantages associated with the liquid state of the oil droplets, the liquid lipid was replaced by a solid lipid, which eventually transformed into solid lipid * For correspondence: V. K. Verma, Tel: vinodmod@yahoo.com nanoparticles (Muller et al.1993; Muller and Lucks 1996). Some of the advantages of SLNs as an alternative particulate carrier are cited in the literature (Muller et al.1993; Muller et al. 1995; ZurMuhlen et al. 1998). Some of them are the small size and relatively narrow size distribution which provide biological opportunities for site specific drug delivery, controlled release of active drug over a long period can be achieved, protection of incorporated drug against chemical degradation, possible sterilization by autoclaving or gammairradiation, no toxic metabolites are produced, avoidance of organic solvent, relatively cheaper and stable, ease of industrial scale production by hot dispersion technique, incorporation of can reduce distinct side effects of drug, eg. Thrombophebitis that is associated with iv. Injection of diazepam or etomidate, surface modification can easily be accomplished and hence can be used for site specific drug delivery system, no biotoxicity of the carrier, incorporation of lipophilic and hydrophilic drugs feasible, high drug payload, and increased drug stability. Many of pharmaceutical researchers have prepared SLNs as an alternative colloidal therapeutic system, utilizing different approaches like modified high shear homogenization and ultra sound techniques, emulsification diffusion method, solvent injection method solvent diffusion method microemulsion method hot homogenization technique. 1136

2 V.K. Verma and A. Ram : Preparation, Characterization and In-vitro release of Piroxiam-loaded Solid Lipid Nanoparticles 1137 Materials and Method Piroxicam was supplied as gift sample by m/s Ramdev pharmaceutical Pvt. Ltd. Thane. Tripalmitin lipid, polyvinyl alcohol (PVAL) and ethyl acetate was purchased from HiMedia Laboratory Pvt. Ltd. Mumbai Maharastra (INDIA). Other chemicals are of analytical grade. Formulation variables and optimization The present investigation was to study the usefulness of tripalmitin as a model lipid for preparation of solid lipid nanoparticles (SLNs) of non- steroidal anti-inflammatory drugs (NSAIDs) piroxicam. Piroxicam loaded tripalmitin solid lipid nanoparticles were prepared by solvent emulsification diffusion technique. The choice and adjustment of the manufacturing parameter are very necessary for the production of solid lipid nanoparticles (SLNs). Various process variables which could effect the preparation and properties of the SLN were identified and studied and the method of preparation accordingly optimized. Various processes variables solubility of the lipids in the water-saturated solvents, stirring rate, influence of temperature, type of solvent, amount of lipid, and amount of stabilizer were selected for optimization of SLNs, preparation methodology. Solubility of the lipids in the water-saturated solvents One of the most important factors that determine the loading capacity of the drug in the lipid is the solubility of lipid in various water-saturated solvent and solubility of drug in melted lipid. In order to determine the solubility of lipid in the different solvents a qualitative trial was performed. The partially water-miscible solvents were saturated with water for five minutes. Approximately 100 mg of lipid were added to 3ml of saturated solvent (3.33% w/v). The samples were sealed and stirred during 12 h. The ability of the solvent to dissolve the lipid was considered when the content appeared transparent on visual observation. In case of lipid insolubility, the temperature required to achieve complete lipid dissolution was determined by heating the vials from 30 to 65 ºC at intervals of 5ºC. In those batches requiring a higher amount of lipid, solubility was previously tested in the same way. Preparation Method: SLN were formed according to a method adopted from Quintanar-Guerrero et al., (1996, 1999a, 1998a, 2005). The solvent ethyl acetate and water were mutually saturated for 5 min at room temperature before use in order to ensure the initial thermodynamic equilibrium of both liquids. When heating was required to solubilize the lipid, the saturation step was performed at this temperature. Typically, 100 mg of lipid and 10 mg drug piroxicam were dissolved in 10 ml of water-saturated solvent and this organic phase (internal phase) was emulsified with 20 ml of the solvent-saturated aqueous solution containing 10% (w/v) of stabilizer (dispersion medium) using a mechanical stirrer at 2600 rpm for 10 min. After formation of an oil in-water emulsion, 80 ml of water (dilution medium) were added to the system in order to allow solvent diffusion into the continuous phase, thus causing the aggregation of the lipid in nanoparticles. When heating was required to dissolve the lipid, both phases were maintained at this temperature and the diffusion step was performed either at room temperature or at the temperature under which the lipid was dissolved. Throughout the process, a constant stirring was maintained Total 27 formulations were prepared on the basis of above variables. The formulation code and respective variables used in the preparation of SLNs. The effect of these variables was observed on particle size, percent yield, drug entrapment efficiency and percent drug loading. Characterization of Solid Lipid Nanoparticles (SLNs) Measurement of size and zeta potential: Size and zeta potential of SLNs were measured in NIPER Mohali Chandigarh by the laser light scattering technique (Malvern Instrument, UK DTS ver.4.10, Serial No. MAL500962). Measurements were obtained at measurement position (nm) 1.05, at a temperature of 25 ºC in disposable sizing cuvette. The scattering intensity data were analyzed by a digital correlator under a unimodal analysis mode. Dispersions were diluted with water to ensure that the light scattering signal, as indicated by the particle counts per second, was within the instrument s sensitivity range. Measurements were made in triplicate for all the batches prepared. The average particle size and zeta potential of optimize formulation batches of piroxicam loaded SLNs were shown in Table 1. Table 1 Qualitative lipid solubility (100 mg) in the different water saturated solvents (3 ml). Lipid (mp C) Methyl ethyl ketone Water-saturated solvent Methyl acetate Ethyl acetate Isopropyl acetate RT MT RT MT RT MT RT MT Tripalmitin (55-60) - 50ºC - 55ºC - 40ºC - 50ºC RT: Room temperature (25 ºC). MT: Minimal temperature for solubilization.

3 1138 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 3 October-December 2010 Scanning Electron Microscopy (SEM): The SLN morphology of some batches was characterized in SAIF Punjab University Chandigarh by SEM after two centrifugations (30,000 rpm/30 min) and resuspension with water. Millipore filter membranes (pore diameter 0.22_m) were used as the carrier medium. Membrane squares (0.5 cm 0.5 cm) were dipped with tweezers in concentrated SLN suspensions and dried at 25 C. Finally, the dried samples and the carrier medium were mounted on stubs and coated with gold (20 nm thickness) using a Sputter Coater JFC-1100 (JEOL, Japan), and were then observed under a JSM-6100 scanning electron microscope (JEOL, Tokyo, Japan). Fig. 1 showed SEM of solid lipid nanoparticles. Transmission electron microscopy (TEM): The morphology of the particles was examined in SAIF Punjab University Chandigarh by TEM (Hitachi 2000 Japan). Samples were obtained from solid lipid nanoparticles containing tripalmitin, drug piroxicam, Stabilizer Polyvinyl alcohol (PVAL), Solvent ethyl acetate and distilled water, stained with a solution of phosphotungustic acid (PTA) at 2% and finely spread over a slab. Fig. 2 showed the morphology of piroxicam loaded solid lipid nanoparticles. Determination of drug entrapment efficiency & Percent drug loading: The precipitate of drug-loaded nanoparticles were dispersed in 80 ml of 1% w/v sodium dodecyl sulfate solution and surged by vortexing for 3 min to dissolve the free drugs. The resulting dispersions were centrifuged for 20 min at 25,000 rpm (REMI Cooling Centrifuge). The drug content in the supernatant after centrifugation was measured by UV Spectroscopy method at 326 nm. The calibration curve of peak area against concentration of piroxicam was y = x at the concentration of piroxicam 4 22 µg/ml (r² = where y = peak area and x piroxicam concentration) the drug entrapment efficiency (Ee) and drug loading (L) of nanoparticles were calculated from Eqs. (1) and (2) (Wa W s) EE (%) = 100..(1) W L (%) = a (Wa W s) 100 (W W + W ) a s L.. (2) Where W a, W s and W L were the weight of drug added in system analyzed weight of drug in supernatant and weight of lipid added in system, respectively. The percent yield of the 9 formulations each batch containing same stabilizer concentration (5%, 10% & 15%), lipid concentration (5%, 10% & 15%) and stirring rate (2200, 2400 & 2600).The effect of these variables on mean percent drug entrapment efficiency, percent drug loading and percent yield are shown in Table 2. Optimized formulations of solid lipid nanoparticles (SLNs) on the basis of effect of different process variables on particle size, percent drug entrapment efficiency and percent drug loading. The four optimized formulations were prepared as presented in Table 3. Differential scanning calorimetry (DSC): The in-vitro drug and lipid interaction was done by differential scanning calorimeter (DSC) (METTLER 305 Switzerland) in CIL, N.I.P.E.R. Mohali. The curve of piroxicam, piroxicam loaded tripalmitin SLNs and unloaded tripalmitin SLNs were recorded on a DSC equipped with a computerized data station. The instrument was calibrated with indium. All samples (about 5mg) were heated in crimped aluminum pans (METTLER 305 Switzerland) at scanning rate of 10ºC mn -1 using dry nitrogen flow (30mL min -1 ). Short- term stability study: The selected SLN formulation was stored at 40 C/75% RH in Newtronic Temperature / Humidity Control Chamber QLH-2004, a period of one month and average particle size and entrapment efficiency were determined. Effect of sterilization: To observe the effect of sterilization on particle size and entrapment efficiency, selected SLN formulation was autoclaved at 121 C for 20 min. In- vitro drug Release study: In-vitro release studies were performed using modified franz diffusion cells having a surface area of cm 2 and 75 ml capacity. Dialysis membrane (LA 401) having pore size 2.4 nm, molecular weight cutoff (HIMEDIA), was used. Membrane was soaked in distilled water for 12 hours before mounting in cell. Piroxicam formulation equivalent to 5 mg of drug was placed in the donor compartment and the recipient compartment was filled with dialysis medium (phosphate buffer of ph 7.4, 75 ml).the content of the cell was stirred with the help of magnetic stirrer at 37 C. At fixed time interval; 1 ml of sample was withdrawn from the receiver compartment through side tube. Fresh phosphate buffer of ph 7.4 was placed to maintain constant volume. Samples were analyzed by UV spectrophotometrically at 326 nm. Statistical analysis: Statistical analysis was performed with SPSS 13.0 software package. Results are expressed as the mean±standard deviation (X±SD). Statistical significance was determined using the Student s t-test and analysis of variance (ANOVA) with p<0.05 as a minimal level of significance.

4 V.K. Verma and A. Ram : Preparation, Characterization and In-vitro release of Piroxiam-loaded Solid Lipid Nanoparticles 1139 Fig. 1 SEM of solid lipid nanoparticles. Fig. 2 Transmission electron microscopy (TEM) of Tripalmitin SLNs (bar = 100nm). Table 2 Optimized formulations Particle Size, Zeta potential & Polydispersity index (PI). S. No. Formulation Code Average Particle Size (nm) Zeta Potential (mv) Polydispersity Index (PI) 1 SLN-B 5 440± ± ± SLN-C ± ± ± SLN- B 8 355± ± ± SLN-C 8 421± ± ±0.020 *mean±sd, n=3

5 1140 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 3 October-December 2010 Table 3 Percent drug entrapment efficiency, percent drug loading and percent yield of SLNs. Process Variables Parameter Mean Percent Drug Entrapment efficiency (%EE) Mean Percent Drug Loading (%L) Mean Percent Yield (%Yield) Stirring Rate ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±4.55 Stabilizer concentration Lipid concentration *mean±sd, n=3 Formulation Code Table 4 Optimized formulations and their selected parameter. Drug: lipid Ratio Parameter Selected Stirring Rate Stabilizer Conc. (%) Temp. (ºC) Average Particle Size Percent Drug Entrapment Efficiency (%EE) Percent Drug Loading (%L) SLN-B 5 1: ± ± ±0.09 SLN-C 5 1: ± ± ±0.11 SLN- B 8 1: ± ± ±0.14 SLN-C 8 1: ± ± ±0.12 *mean ±SD, n=3 Results and Discussion Solubility of the lipids in the water-saturated solvents Solubility studies (Table 1) indicates that amongst the ethyl methyl keton, methyl acetate, ethyl acetate and isopropyl acetate, ethyl acetate effectively solubilized lipid tripalmitin. The solubilizing potential coupled with already reported biocompatibility and acceptability of lipid tripalmitin for topical and parental route has favored its selection for the present study. Particle size and Zeta potential The particle size, polydispersity index and zeta potential of colloidal carriers are important characteristics of SLNs from which the solubility of drug-loaded SLNs can be predicted. Average particles size, polydispersity index and zeta potential of selected formulation are shown in Table 2. All the formulations had shown in nanosize range ( ) with narrow size distribution (polydispersity index= ). Besides production parameter concentration of lipid, stabilizer concentration, stirring rate, influence of temperature and solubility of lipid in different solvents influence the outcomes of the procedure. Leaving all other parameter constant, in this study the variables was composition of lipid, stirring rate and stabilizer concentration in alternatively varying from 5, 10 & 15%, 2200, 2400 & 2600 rpm & 5, 10 & 15% (Table 3) respectively. The relationship between the amounts of lipid and the SLN size is shown in Table 4. Result revealed that Low concentrations of lipid allow production of SLN as small as approximately 100 nm. Particle size increased with higher lipid amounts. This effect was very important between 5, 10 and 15% lipid concentration, above these percentages, microparticles were always obtained. Moreover, if we want to prepare SLN with high lipid concentrations, other process variables (e.g., stabilizer concentration and/or temperature) need to be modified. The fact that SLN formation is highly dependent on lipid concentration can be explained in terms of the tendency of lipid aggregates (generated during diffusion) to coalesce at high lipid concentrations. Furthermore, the high viscosity and low limiting concentration for lipid aggregation at the

6 V.K. Verma and A. Ram : Preparation, Characterization and In-vitro release of Piroxiam-loaded Solid Lipid Nanoparticles 1141 interface will cause a decrease of solvent diffusion and hence fewer lipid molecules will be carried into the aqueous phase. Therefore, the formation and stabilization of small lipid aggregates at these concentrations are reduced (Quintanar-Guerrero et al., 1999b). The choice of the stabilizer and their concentration is great impact on the quality of SLNs dispersion. Investigating the influence of the stabilizer concentration on the particle size of SLNs dispersions, we obtain best result with 15% stabilizer (polyvinayl alcohol). High concentrations of the stabilizer reduce the surface tension and facilitate the particle partition. The decrease in particle size is connected with a tremendous increase in surface area. The measurement of the zeta potential allows prediction about the stability of colloidal aqueous dispersions. Usually, particle aggregation is less likely to occur for changed particle with high zeta potential due to electric repulsion. In general lipid nanoparticles are negatively charged on the surface. The determination of zeta potential was performed in aqueous SLNs stored at room temperature. Scanning Electron Microscopy (SEM) SEM image of the formulations were derived from JSM scanning electron microscope (JEOL, Tokyo, Japan) has been presented in Fig.1. SEM confirms that the SLNs of selected formulations are circular in shape. They are smooth and well separated on the surface. Transmission electron microscopy (TEM) TEM image of the formulations were derived from TEM (Hitachi 2000 Japan) has been presented in Fig.2. TEM confirms that the SLNs of selected formulations are circular in shape and particles are nanometeric size. Drug entrapment efficiency and Percent drug loading According to Professor Muller the prerequisite to obtain a sufficient loading capacity was a sufficiently high solubility of the drug in the lipid. Relatively higher drug EE% was one of the major advantages of SLNs. Table 4 data showed the drug EE% of selected optimized formulation. The loading capacity of SLN was found to be satisfactory high. The data showed that drug entrapment efficiency and loading capacity of nanoparticles were increased from 83.74±0.87 to 85.61±0.93% and from 7.70±0.09 to 7.82±0.05%, respectively by increasing the lipid concentration. For SLN formulations, the entrapment efficiency is lower for the sample with lower lipid concentration. It has to be noticed that during the cooling process, the lipid solidifies and the drug is distributed in to the shell of the particles, if the concentration of the drug in the melted lipid is well below its saturation solubility. Differential scanning calorimetry (DSC) The thermal curve of drug unloaded tripalmitin lipid and piroxicam loaded tripalmitin lipid nanoparticles showed endothermic peaks at C and C respectively (Fig. 3). The melting endothermic peaks of the drug loaded nanoparticles appeared at slightly lower temperature. The decrease in melting temperature of nanoparticle formulated piroxicam loaded tripalmitin lipid compare with the unloaded lipid has been attributing to their small size and presence of stabilizers. Short- term stability study After one month storage at 40 C/75% RH, it has been found that the particle size of piroxicam loaded SLNs increases by 8nm and entrapment efficiency was lowered by 2.68% shown in Table 5. Transition of dispersed lipid from metastable forms to stable form might occur slowly on storage due to small particle size and the presence of emulsifiers and stabilizers that may lead to drug expulsion from solid lipid nanoparticles. Therefore lowered entrapment efficiency observed on storage may be due to expulsion during lipid modification. Effect of sterilization Effect of sterilization on particle size and entrapment efficiency was shown in Table 6 in selected formulation, size of particle increases almost two times after sterilization, but still they are in nanosize. It was found that sterilization by autoclaving has least effect on entrapment efficiency. Therefore, sterilization by autoclaving can performed for solid lipid nanopaarticles of tripalmitin stabilized with polyvinyl alcohol. In- vitro release study Many research groups used vertical or flow-through franz diffusion cell and dialysis bag/tubes for the study of drug release from solid lipid and polymeric nanoparticles and niosomes. In order to evaluate the controlled release potential of the investigated formulations, the diffusion of piroxicam from the tripalmitin lipid particle was investigated over 24 h. each sample was analyzed in triplicate. The results are shown in Table 7 and Fig. 4 the release rate of piroxicam depends on the total concentration of piroxicam present in the formulation. Piroxicam release more quickly when using lower concentration because of the drug-enriched shell model proposed for these particles (piroxicam loaded SLNs). Due to the large drug loading in SLNs the degree of diffusion can be decreased.

7 1142 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 3 October-December 2010 Table 5 Effect of time of storage (at 40 C/75% RH) on particle size and entrapment efficiency. Formulation code Particle size (nm) Entrapment efficiency (%) Zero day One month Zero day One month SLN-B 5 440±13 448± ± ±0.80 SLN-C 5 420±17 429± ± ±0.85 SLN- B 8 355±22 367± ± ±0.55 SLN-C 8 421±16 430± ± ±0.67 *mean ±SD, n=3 Table 6 Effect of sterilization on particle size and entrapment efficiency Formulation code Particle size (nm) Entrapment efficiency (%) Before After Before After SLN-B 5 440±13 782± ± ±0.66 SLN-C 5 420±17 728± ± ±0.67 SLN- B 8 355±22 698± ± ±0.78 SLN-C 8 421±16 778± ± ±0.75 *mean ±SD, n=3 Table 7 In vitro Percent drug release of Piroxicam from SLNs S.No. Time Duration (Hours) Percent Drug Release (%) SLNs Formulation Code SLN-B5 H SLN-C5 H SLN-B8 H SLN-C8 H ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.22 Mean ±S.D. (n=3)

8 V.K. Verma and A. Ram : Preparation, Characterization and In-vitro release of Piroxiam-loaded Solid Lipid Nanoparticles 1143 Percent Drug Release (%) Percent Drug Release (%) Time (Hour) SLN-B5 H SLN-C5 H SLN-B8 H SLN-C8 H Fig. 4 In-vitro Percent Drug Release of Piroxicam from SLNs. Percentage of piroxicam release from SLNs up to 24 h is shown in Table 6 and Fig. 4 for selected formulations. The release pattern revealed that there was an initially burst effect followed by a prolong release of drug. This is because the drug may be located primarily in the shell of the particles. Other factor contributing to a fast release are large surface area, high diffusion coefficient (small molecular size), low matrix viscosity and short diffusion distance of the drug. The drug enriched core is surrounded by a drug free lipid shell. Due to the increased diffusional distance and hindering effects by surrounding solid lipid shell, the drug has a sustained release profile. The formulations were further subjected to release kinetic studies. The release data was fitted into first order and Higuchi equations. Release of drug from almost all the SLNs followed Higuchi equation better than first order equation. Conclusion Solid lipid nanoparticles represent a particular system, which can be produce with an established technique, solvent emulsification diffusion process allowing production on industrial scale. It can be achieved after the selection of optimal formulation and process parameters. The size distribution of SLNs revealed a mono-dispersion profile in distilled water. In vitro release of piroxicam from SLNs in the phosphate buffer of ph 7.4, exhibited a biphasic pattern with an initial burst and prolonged release over 24 h. References Al-Kindy SMZ, Al-Wishahi A, Eldin F, Suliman O. A sequential injection method for the determination of piroxicam in pharmaceutical formulations using europium sensitized fluorescence, 12th Int. Conference On Flow Injection Analysis, 64, (2004). Almeida AJ, Runge S, Muller RH. Peptide-loaded solid lipid nanoparticles (SLN) influence of production parameters, Int. J. Pharm., 149, (1997). Alvarez-Figueroa MJ, Blanco-Mendeza J. Transdermal delivery of methotrexate: iontophoretic delivery from hydrogels and passive delivery from Microemulsion, Int. J. Pharm., 215, (2001). Bekerman T, Golenser J, Domb A. Ciclosporin nanoparticulate lipospheres for oral administration, J. Pharm. Sci. 93, (2003). Bocca C, Caputo O, Cavalli R, Gabriel L. Phagocytic uptake of fluorescent stealth and non-stealth solid lipid nanoparticles, Int. J. Pharm., 175, (1998). Borgia L S, Regehly M, Sivaramakrishnan R, Mehnert W, Korting HC, Danker K, Roder B, Kramer KD, Schfer-Korting M. Lipid nanoparticles for skin penetration enhancement correlation to drug localization within the particle matrix as determined by fluorescence and parelectric spectroscopy, J. Controlled Release, 110, (2005). Casadei MA, Cerreto Cesa S, Feeney MGM, Marianecci C, Paolicell P. Solid lipid nanoparticles incorporated in dextran hydrogels: A new drug delivery system for oral formulation, Int. J. Pharm. (In Press) (2006).

9 1144 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 3 October-December 2010 Castelli F, Puglia C, Sarpietro MG, Rizza L, Bonina F. Characterization of indomethacin-loaded lipid nanoparticles by differential scanning calorimetry, Int. J. Pharm., 304, (2005). Cavalli R, Caputo O, Carlotti ME, Trotta M, Scarnecchia C, Gasco MR. Sterilization and freeze-drying of drug-free and drug-loaded solid lipid nanoparticles, Int. J. Pharm. 148, (1997). Cavalli R, Caputo O, Gasco MR. Preparation and characterization of solid lipid nanospheres containing Paclitaxel, Eur. J. Pharm. 10, (2000). Cavalli R, Peira E, Caputo O, Gasco MR. Solid lipid nanoparticles as carriers of hydrocortisone and progesterone complexes with beta-cyclodextrins, Int. J. Pharm., 182, (1999). Charcosset C, El-Harati A, Fessi H. Preparation of solid lipid nanoparticles using a membrane contactor, J. Controlled Release, 108, (2005). Chen H, Chang X, Du D, Liu W, Liu J, Weng T, Yang Y, Xu H, Yang X. Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting., J.Controlled Release, 110, (2006). Chen ZMH, Weng T, Yang Y, Yang X. Solid lipid nanoparticle and microemulsion for topical delivery of triptolide, Eur. J. Pharm. and Biopharm., 56, (2003). Colombo AP, Briancon S, Lieto J, Fessi H. Project design and use of a pilot plant for nanocapsule production, Drug Dev. Ind. Pharm. 27, (2001). Cortesi R, Esposito E, Luca G, Nastruzzi, C. Production of lipospheres as carriers for bioactive compounds, Biomaterials 23, (2002). El-Ries MA. Spectrophotometric Determination of Piroxicam and Tenoxicam in Pharmaceutical Preparations Using Uranyl Acetate as a Chromogenic Agent., Analytical Letters, 31, (2004). Els-Shabouri MH. Positively charged nanoparticles for improving the oral bioavailability of cyclosporin-a, Int. J. Pharm., 249, (2002). Florey K. Analytical profile of drug substances, Academic Press, Vol. 15, 509 (1979). Freitas C, Muller RH. Effect of light and temperature on zeta potential and physical stability in solid lipid nanoparticle (SLNs) dispersions, Int. J. Pharm., 168, (1998). Fuentes MG, Prego C, Torres D, Alonso MJ. A comparative study of the potential of solid triglyceride nanostructures coated with chitosan or poly (ethylene glycol) as carriers for oral calcitonin delivery, Eur. J. Pharm. Sci. 25, (2005). Fuentes MG, Torres D, Alonso MJ. New surface-modified lipid nanoparticles as delivery vehicles for salmon calcitonin, Int. J. Pharm. 296, (2005). Garcia-Fuentes M, Torres D, Alonso MJ. Design of lipid nanoparticles for the oral delivery of hydrophilic macromolecules, Coll. Surf.B. Biointerfac., 27, (2002). Gasco MR. Method for producing solid lipid microspheres having a narrow distribution, US Patent No. 5,250,236 (1993). Goppert TG, Muller RM. Adsorption kinetics of plasma proteins on solid lipid nanoparticles for drug targeting, Int. J. Pharm. 302, (2005). Goppert TM, Muller RH. Protein adsorption patterns on poloxamer- and poloxamine-stabilized solid lipid nanoparticles (SLN), Eur. J.Pharm. and Biopharm., 60, (2005). Heiati H, Phillips NC, Tawashi R. Evidence for phospholipid bilayer formation in solid lipid nanoparticles formulated with phospholipid and triglyceride, Pharm. Research., Vol. 13, No.2 (1996). Houa DZ, Xieb CS, Huangb K, Zhuc, CH. The production and characteristics of solid lipid nanoparticles (SLNs), Biomaterials, 24, (2003). Hu FQ, Jiang SP, Du YZ, Yuan H, Ye YQ, Zeng S. Preparation and characteristics of monostearin nanostructured lipid carriers, Int. J. Pharm., 314, (2006). Hu FQ, Jiang SP, Du YZ, Yuan H, Ye, YO, Zeng S. Preparation and characterization of stearic acid nanostructured lipidcarriers by solvent diffusion method in an aqueous system, Colld. Surf. B. Biointerfac, 45, (2005). Hu FQ, Yuan H, Zhang HH, Fang M. Preparation of solid lipid nanoparticles with clobetasol propionate by a novel solvent diffusion method in aqueous system and physicochemical characterization, Int. J. Pharm. 239, (2002). Indian Pharmacopeia, Govt. of India, Ministry of health and family welfare, Published by the controller of Publications, Delhi. Vol., I II, Jee JP, Lim SJ, Park JS, Kim CK. Stabilization of all-trans retinol by loading lipophilic antioxidants in solid lipid nanoparticles, Eur. J.Pharm. and Biopharm.,63, (2006). Jie L, Wen Hu., Huabing Chena Qian N, Huibi Xu, Xiangliang Y. Isotretinoin-loaded solid lipid nanoparticles with skin targeting for topical delivery, Int. J. Pharm., (2006). Kenon AP, Schawartz M A. Drug stability, J. Am. Chem. Soc., 60, (1983).

10 V.K. Verma and A. Ram : Preparation, Characterization and In-vitro release of Piroxiam-loaded Solid Lipid Nanoparticles 1145 Kipp JK. The role of solid nanoparticle technology in the parenteral delivery of poorly water-soluble drugs, Int. J. Pharm. 284, (2004). Kwon HY, Lee JH, Choi SW, Jang Y, Kim JH. Preparation of PLGA nanoparticles containing estrogen by emulsification diffusion method, Colloid Surf. A 182, (2001). Lachman L, Lieberman HA, Kanig LJ. The Theory and Practice of Industrial Pharmacy, Varghese Publishing House Bombay, Lamprecht A, Saumet JL. Lipid nanocarriers as drug delivery system for ibuprofen in pain treatment, Int. J. Pharm. 278, (2004). Li Y, Dong L, Chang AJX, Xue H. Preparation and characterization of solid lipid nanoparticles loaded traditional chinese medicine, Int. J. Biological Macromolecules, 38, (2006). Lippacher A, Muller RH, Mader K. Semisolid SLNs dispersions for topical application: influence of formulation and production parameters on viscoelastic properties, Eur.J.Pharm.Biopharm., 53, (2002). Lippachera A, Muller RH, Mader K. Liquid and semisolid SLNe dispersions for topical application rheological characterization, Eur. J.Pharm. and Biopharm., 58, (2004). Maia CS, Mehnert W, Korting MHF. Solid lipid nanoparticles as drug carriers for topical glucocorticoids, Int. J. Pharm., 196, (2000). Marengo Emilio, Cavalli Roberta, Caputo Otto, Rodriguez Lorenzo, Gasco Maria Rosa. Scale-up of the preparation process of solid lipid nanospheres Part I, Int. J. Pharm., 205, 3 13 (2000). Mehnert W, Mader K. Solid lipid nanoparticles Production, characterization and applications, Adv. Drug Del. Rev., 47, (2001). Meia Z, Lia X, Wua Q, Hua S, Yang X. The research on the anti-inflammatory activity and hepatotoxicity of triptolideloaded solid lipid nanoparticles, Pharmacol. Res., 51, (2005). Miglietta A, Cavalli R, Bocca C, Gabriel L, Gasco MR. Cellular uptake and cytotoxicity of solid lipid nanospheres (SLN) incorporating doxorubicin or paclitaxel, Int. J. Pharm., 210, (2000). Morel S, Ugazio E, Cavalli R, Gasco MR. Thymopentin in solid lipid nanoparticles, Int. J. Pharm., 132, (1996). Morela S, Terrenob E, Ugazioa E, Aimeb S, Gascoa MR. NMR relaxometric investigations of solid lipi nanoparticles (SLN) containing gadolinium (III) complexes, Eur. J. Pharm. Biopharm., 45, (1998). Muller RH, Maeder K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug delivery a review of the state of the art, Eur. J.Pharm. and Biopharm., 50, (2000). Muller RH, Radtke M, Wissing SA. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations, Adv. Drug Deliv. Rev. 54 (1), S131 S155 (2002). Muller RH, Rungea S, Ravelli V, Mehnert W, Thunemannc AF, Souto EB. Oral bioavailability of cyclosporine: Solid Lipid Nanoparticles (SLNs) versus drug nanocrystals, Int. J. Pharm. 317, (2006). Olbrich C, Gessner A, Kayser O, Muller RH. Lipid-drug conjugate (LDC) nanoparticles as novel carrier system for hydrophilic antitrypanosomal drug diminazenediaceturate, J. Drug Target. 10, (2002). Norouzi MR, Ganjali S, Labbafi A Mohammadi. Subsecond FFTadsorptive Voltammetric Technique as a Novel Method for Subnano Level Monitoring of Piroxicam in its Tablets and Bulk Form at Au Microelectrode in Flowing Solutions, Analytical Letters, 40, (2007). Pandey R, Sharma S, Khuller GK. Tuberculosis: Oral solid lipid nanoparticle-based antitubercular chemotherapy, Tuberculosis. 85, (2005). Quintanar-Guerrero D, All emann E, Doelker E, Fessi H. A mechanistic study of the formation of polymer nanoparticles by the emulsification-diffusion technique,colloid. Polym. Sci. 275, (1997). Quintanar-Guerrero D, All emann E, Fessi H., Doelker E. Preparation techniques and mechanisms of formation of biodegradable nanoparticles from preformed polymers, Drug Dev. Ind. Pharm. 24, (1998a). Quintanar-Guerrero D, Allemann E, Fessi H, Doelker E. Pseudolatex preparation using a novel emulsion-diffudion process involving direct displacement of partially watermiscible solvents by distillation, Int. J. Pharm. 188, (1999b). Quintanar-Guerrero D, Fessi H, Allemann E, Doelker E. Influence of stabilizing agents and preparative variables on the formation of poly (d,l-lactic acid) nanoparticles by an emulsification-diffusion technique, Int. J. Pharm. 143, (1996). Quintanar-Guerrero D, Ganem-Quintanar A, Allemann E, Fessi H, Doelker E. Influence of the stabilizer coating layer on the purification and freeze-drying of poly(d,l-lactic acid) nanoparticles prepared by an emulsion-diffusion technique, J. Microencapsul. 15, (1998b). Quintanar-Guerrero D, Gurny R, Allemann E, Fessi H, Doelker, E. Method for producing aqueous colloidal dispersions of nanoparticles. WO Patent No. 01,002,087 (1999a).

11 1146 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 3 October-December 2010 Reddy H, Murthy RSR. Etoposide-Loaded Nanoparticles Made from Glyceride Lipids: Formulation,Characterization, in Vitro Drug Release, and Stability Evaluation, AAPS, Pharm.Sci.Tech., 6(2), Article 24 (2005). Saupe A, Gordon CK, Rades T. Structural investigations on nanoemulsions, solid lipid nanoparticles and nanostructured lipid carriers by cryo-field emission scanning electron microscopy and Raman spectroscopy, Int. J. Pharm., 314, (2006). Savaer AA, Karata Y, Ozkan N, Yüksel S A, Ozkan T Baykara. Validated LC Determination of the Piroxicam-ß-Cyclodextrin Inclusion Complex in Tablets and in Human Plasma, Chromatographia, 59, (2004). Schubert MA, Harms M, Muller-Goyman CC. Structural investigations on lipid nanoparticles containing high amounts of lecithin, Eur. J. pharm. Sci., 2 7, (2006). Schubert MA, Muller-Goymann CC. Characterisation of surfacemodified solid lipid nanoparticles (SLN): Influence of lecithin and nonionic emulsifier, Eur. J.Pharm. and Biopharm., 61, (2005). Shahgaldian P, Da Silva E, Coleman AW, Rather B, Zaworoto MJ. Para-acyl-calyx-arene based solid lipid nanoparticles (SLNs) a detailed study of preparation and stability parameters, Int. J. Pharm. 253, (2003). Söderberg L, Dyhre H, Roth B, Bjorkman S. The inverted cup A novel in vitro release technique for drugs in lipid formulations, J. Control. Release, 113, (2006). Souto, EB, Wissing SA, Barbosa CM, Muller RH. Development of a controlled release formulation based on SLN and NLC for topical clotrimazole delivery, Int. J. Pharm., 278, (2004). Soutoa EB, Wissinga SA, Barbosab CM, Muller RH. Evaluation of the physical stability of SLN and NLC before and after incorporation into hydrogel formulations, Eur. J.Pharm. and Biopharm., 58, (2004). Tabatta K, Sametib M, Olbricha C, Muller RH, Lehrb CM. Effect of cationic lipid and matrix lipid composition on solid lipid nanoparticle-mediated gene transfer, Eur. J. Pharm. and Biopharm., 57, (2004). The Merck Index. An Encyclopedia of Chemicals, Drug and Biologicals, 12 th edition, Merch Research Laboratories, Division of Merck and Co., Inc., 7661, Trotta M, Debernardi F, Caputo O. Preparation of solid lipid nanoparticles by a solvent emulsification diffusion technique, Int. J. Pharm. 257, (2003). Venkateswarlu V, Manjunath K. Preparation, characterization and in vitro release kinetics of clozapine solid lipid nanoparticles, J, Controlled Release, 95, (2004). Vyas SP, Khar RK, Controlled drug delivery concepts and advances, First edition, Vallabh Prakashan Delhi, (2002). Westese, K, Bunjes H, Koch MHJ. Physicochemical characterization of lipid nanoparticles and evaluation of their loading capacity and sustained release potential, J, Controlled Release, 48, (1997). Westesen K, Siekmann B, Koch MHJ. Investigations on the physical state of lipid nanoparticles by synchroton radiation X-ray diffraction, Int. J. Pharm. 93, (1993). Wissing SA, Kayser O, Muller RH. Solid lipid nanoparticles for parenteral drug delivery, Adv. Drug Deliv. Rev. 56, (2004). Yuan Y, Li S, Feng-kui Mo F, Zhong D. Investigation of microemulsion system for transdermaldelivery of meloxicam, Int. J. Pharm., (In Press) (2006). Zhang N, Ping Q, Huang G, Xu W, Cheng Y, Han X. Lectin modified Solid Lipid Nanoparticles as Carriers for Oral Administration of Insulin, Int. J. Pharm., (In Press) (2006). Zur Muhlen, A, Schwarz C, Mehnert W. Solid lipid nanoparticles (SLN) for controlled drug delivery- Drug release and release mechanism, Eur. J. Pharm. Biopharm, 45, (1998).